Nucleotide and deduced amino acid sequences of tumor gene Int6

ABSTRACT

The present invention discloses the isolation of the human and murine wild-type Int6 gene and the cDNAs sorresponding to these genes. The invention further describes the use of reagents derived from the nucleic acid and amino acid sequences of the Int6 gene in diagnostic methods, immunotherapy, gene therapy and as vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 09/858,152, filed May14, 2001, now U.S. Pat. No. 6,737,251, which is a division of U.S.application Ser. No. 09/378,842, filed Aug. 23, 1999, now issued as U.S.Pat. No. 6,342,392, which is a divisional of U.S. application Ser. No.08/875,847, filed Sep. 25, 1997, now issued as U.S. Pat. No. 6,255,105,which is the national stage under § 371 of PCT Application No.PCT/US96/01884, filed Feb. 9, 1996, published in English under PCTArticle 21(2). PCT Application No. PCT/US96/01884 is acontinuation-in-part of U.S. application Ser. No. 08/385,998, filed Feb.9, 1995, now abandoned.

FIELD OF INVENTION

The present invention relates to the area of cancer diagnostics andtherapeutics. More specifically, the invention relates to the Int6 geneand to the use of reagents derived from the nucleic acid and deducedamino acid sequences of the Int6 gene in gene therapy, vaccines,diagnostic methods and immunotherapy.

BACKGROUND OF INVENTION

The mouse mammary tumor virus (MMTV) is a retrovirus which has beenshown to act as an insertional mutagen that causes the deregulation ofexpression of cellular genes adjacent to the site of MMTV integration inmammary tumors (Varmus, H. E. (1982). Cancer Surv., 1:309–320). Miceinfected with MMTV frequently develop preneoplastic hyperplasticalveolar nodules (HAN) (Daniel, C., et al. Proc. Natl. Acad. Sci. USA.,61:53–60; DeOme, K. B., (1959) J. Natl. Cancer Inst., 78:751–757;Medina, D., (1973) Methods Cancer Res., 7:3–53; Smith, G., et al. (1984)Cancer Res., 44:3426–3437) and the passage of these nodules in clearedmammary fat pads of syngeneic mice by serial outgrowth often results inthe development of mammary tumors within these mice in a stochasticmanner. In addition, it is not uncommon to find metastatic lesions inthe lungs of mice bearing outgrowths with mammary tumors. For thesereasons, there is considerable interest in identifying MMTV-inducedmutational events that may contribute to different stages of tumordevelopment.

Using the MMTV genome as a molecular tag, five loci (Wnt-1/Int-1,Fgf-3/Int-2, Int-3, Wnt-3, and Fgf-4/Hst/k-FGF) have been identifiedwhich represent common integration sites (designated Int loci) for MMTVin mouse mammary tumors (Dickson, C., et al. (1984) Cell., 37:529–536;Gallahan, D., et al. (1987) J. Virol., 61:218–220; Nusse, R., et al.(1982) Cell., 31:99–109; Peters, G., et al. (1989) Proc. Natl. Acad.Sci. USA., 86:5678–5682). Transgenic mouse studies utilizing transgenesin which the MMTV LTR was linked to either the WNT-1, Fgf-3, or Int-3genes have demonstrated that activation of expression of these genescontributes to mammary tumor-1-genesis (Jhappan, C., et al. (1992) Genes& Develop., 6:345–355; Muller, W. J., et al. (1990) Embo J., 9:907–913;Tsukamoto, A. S., et al. (1988) Cell., 55:619–625).

The present invention describes the isolation of a new Int genedesignated Int6 and the use of the gene, its gene product, and reagentsderived from the gene and its gene product, in diagnostic methods,vaccines, immunotherapy and gene therapy.

SUMMARY OF INVENTION

The present invention relates to the isolation of the Int6 gene. Theinvention also relates to the murine and human cDNAs which comprise thecoding sequence of the Int6 gene. The invention further relates tonucleic acid sequences derived from the Int6 gene and the Int6 cDNAs andthe use of these nucleic acid sequences as probes to isolate homologuesof the Int6 gene in other mammals or as probes to detect mutations ofthe Int6 gene.

It is also an object of the present invention to provide syntheticnucleic acid sequences capable of directing production of recombinantInt6 protein and peptide fragments derived therefrom, as well asequivalent natural nucleic acid sequences. Such natural nucleic acidsequences may be isolated from a cDNA or genomic library from which thegene capable of directing the synthesis of the Int6 protein may beidentified and isolated. For the purposes of this application, nucleicacid sequence refer to RNA, DNA, cDNA or any synthetic variant thereof.

The present invention further relates to Int6 protein and peptidesderived therefrom.

The invention also relates to antibodies directed against Int6 proteinor peptides derived therefrom.

The invention also provides methods for detecting mutations of Int6 genewhere detection of such mutations is useful in determining the presenceof a neoplastic tissue in a subject or a genetic predisposition tocancer in a subject.

A first method for detecting mutations of the Int6 gene comprisesanalyzing DNA of a subject for mutations of the Int6 gene.

A second method for detecting mutations of the Int6 gene comprisesanalyzing RNA of a subject for alterations in the Int6 mRNA expression.

Yet another method for detecting mutations of the Int6 gene comprisesanalyzing protein of a subject for alterations in Int6 proteinexpression.

The present invention also provides pharmaceutical compositions for useas vaccines for immunizing a subject against cancer and for use inimmunotherapy methods. One such composition comprises nucleic acidsequence capable of directing host organism synthesis of Int6 protein orpeptide fragments thereof while a second pharmaceutical compositioncomprises Int6 protein or peptides derived therefrom.

The above pharmaceutical compositions may also be used in immunotherapymethods for treating a subject having cancer. For use in immunotherapy,the present invention further provides a third pharmaceuticalcomposition comprising antibodies directed against Int6 protein orpeptides derived therefrom where such antibodies are coupled to toxinmolecules, radioisotopes or drugs.

The present invention therefore relates to application of immunotherapyto subjects having cancer comprising administering one or more of theabove pharmaceutical compositions to said subject in a therapeuticallyeffective amount.

The invention further relates to a method for treating a subject havingcancer comprising:

-   -   (a) immunizing the subject with an amount of an expression        vector encoding Int6 protein or with Int6 protein itself, said        amount effective to elicit a specific T cell response;    -   (b) isolating said T cells from said immunized subject; and    -   (c) administering said T cells to said immunized subject or to        an unimmunized subject in a therapeutically effective amount.

The invention also provides a diagnostic kit for determining thenucleotide sequence of Int6 alleles by the polymerase chain reaction,said kit comprising purified and isolated nucleic acid sequences usefulas PCR primers. These PCR primers are also useful in analyzing DNA orRNA of a subject for mutations of the Int6 gene.

The present invention further provides a method for supplying thewild-type Int6 gene to a cell having altered expression of the Int6protein by virtue of a mutation in the Int6 gene, the method comprising:introducing a wild-type Int6 gene into a cell having altered expressionof the Int6 protein such that said wild-type gene is expressed in thecell.

DESCRIPTION OF FIGURES

FIGS. 1A and 1B show the results of Southern blot analyses in which 10micrograms of cellular DNA was digested by EcoRI, separated by agarosegel electrophoresis, and hybridized with MMTV LTR probe. In FIG. 1A, theDNAs analyzed were isolated from a CZZ-1 pre-neoplastic hyperplasticoutgrowth line designated CZZ-1 HOG (lane 1) and from CZZ-1 derivedmammary tumors 22 (lane 2), 1262 (lane 3), 1263 (lane 4), 20 (lane 5),19 (lane 6), 23 (lane 7), 21 (lane 8) 24 (lane 9), 8 (lane 10), 9 (lane11), 12 (lane 12), 13 (lane 13) and 14 (lane 14) as indicated at the topof FIG. 1A. In FIG. 1B, the DNAs analyzed were isolated from CZZ-1derived tumor 4973 (lane 1) and from 11 independent lung metastasis(lanes 2–12) from a mouse bearing tumor 4973 as indicated at the top ofFIG. 1B.

FIGS. 2A–2C show the results of Southern blot analyses of 10 microgramsof cellular DNA digested with EcoRI, separated by agarose gelelectrophoresis and hybridized as follows. Lanes 1 and 2 of the blots inFIGS. 2A and 2B were hybridized with probe D which corresponds to hostflanking sequences. Lane 2 of the blots in FIGS. 2A and 2B wassubsequently hybridized with MMTV gag sequences (a 2.0 kb-Pst-XhoIfragment from MMTV GR-40; Gallahan and Callahan J. Virol, 61, 66–74:(1987)) and the results are shown in lane 3 in FIGS. 2A and 2B. Lanes 1and 2 in FIG. 2C were hybridized with probe C. Lane 2 was subsequentlyhybridized with MMTV env sequences (1.7 kb PstI fragment from MMTV(C3H); Gallahan and Callahan J. Virol, 61: 66–74; (1987)) and the resultis shown in lane 3 in FIG. 2C. The DNAs analyzed were isolated fromCZZ-1 HOG derived tumor 22 (FIG. 2A, lanes 2 and 3), independent mammarytumor 1139 (FIG. 2B, lanes 2 and 3) independent mammary tumor 3134 (FIG.2C, lanes 2 and 3), and normal liver. (Lane 1 in FIGS. 2A–2C). Thepresence of the arrow in FIGS. 2A–2C indicates the site of the MMTVinduced rearranged restriction fragment. The locations of probes C and Din the Int6 gene are shown in FIG. 3.

FIG. 3 is a schematic diagram of the murine Int6 locus in which thelocation of the four overlapping lambda clones which span the Int6 locus(designated 1 to 4) are shown relative to a partial restriction map ofsites for EcoRI (E), XbaI (X), PstI (P), BglII (BGL), HindIII (H), andBamHI (B) in the Int6 locus. The location of the Int6 exons and ofrestriction fragments used as probes A–D are indicated below therestriction map by solid and hatched boxes respectively. The scale ofthe restriction map is given in increments of 1.0 kb at the bottom ofthe Figure while the location and transcriptional orientation ofintegrated MMTV proviral genomes within the Int6 gene of tumors 1139 and3144, or within the Int6 gene of the CZZ-1 HOG, is indicated byarrowheads above the restriction map.

FIG. 4 shows the results of Northern blot analysis of total RNA isolatedfrom Int6 negative tumor (tumor 178) and the Int6 positive tumors (22and 1139). The RNAs were denatured in the presence of formaldehyde,separated on a 1% agarose gel containing formaldehyde, and hybridizedwith probe A.

FIG. 5 shows the complete nucleotide sequence of the murine 1.4 kb Int6cDNA (SEQ ID NO: 1) where intron breaks are indicated by small arrows(▴) above the start of the next exon and the deduced amino acid sequenceof the gene product is given below the nucleotide sequence. Potentialphosphorylation sites for cyclic AMP/cyclic GMP-dependent protein kinase(∘), protein kinase C (Δ), tyrosine kinases (−), casein kinase II (⋄),and glycosylation sites (□) are indicated in the deduced amino acidsequence. Abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp;E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; M. Met

FIG. 6 shows the results of a “Zoo” blot in which cellular DNA (10 μgeach) isolated from C. Elegans, Drosophila, Xenopus, chicken, mouse andhuman was hybridized with Int6 cDNA under high stringency conditions.

FIG. 7 shows the results of Northern blot analyses of total RNA (10 μgeach) isolated from normal adult tissues (top panel) and from developingembryos (lower panel). The RNAs were denatured in the presence offormaldehyde, run on 1% agarose gels containing formaldehyde and thentransferred to a nylon membrane and hybridized sequentially with aβ-actin-probe (the 2.3 kb mRNAs) and then a murine Int6 cDNA probe (the1.4 kb mRNAs).

FIG. 8 shows a schematic illustrating the location and nucleotidesequence of a cryptic transcription stop signal (underlined) in thereverse sequence of the MMTV LTR where the sequence shown corresponds tobases 464 through 497 from the three prime end of the LTR. The hatchedbox corresponds to an Int6 exon and the open box corresponds to the LTRof an integrated MMTV proviral genome integrated within Int6. The U5, Rand U3 regions of the MMTV LTR are indicated as well as thetranscriptional orientations of the MMTV genome and the Int6 gene.

FIGS. 9A and 9B show the nucleotide sequences of the junctions betweenMMTV and Int6 sequences in chimeric Int6-MMTV LTR RNA species detectedin tumors 1139 (FIG. 9A) and 22 (FIG. 9B). In FIG. 9A, the nucleotidesequence shown (SEQ ID NO: 33) begins at the 5′ end of exon with thenucleotide sequence shown in lower case letters corresponding to intro 5sequences (nucleotides 148–212 of SEQ ID NO: 33). Amino acid sequencesin RNA species 2 which are identical to those in RNA species 1 areindicated by dots (see amino acids 60–108 of SEQ ID NO: 4), andnucleotide sequence which have been spliced out in RNA species 2 areindicated by dashes (see nucleotides 148–213 of SEQ ID NO: 33).Nucleotide and amino acid sequences which are underlined are from theintegrated MMTV genome (nucleotides 214–687 of SEQ ID NO: 33 and SEQ IDNO: 34. respectively).

In FIG. 9B, the nucleotide sequence shown (SEQ ID NO: 35) for thechimeric RNA species detected in tumor 22 begins at the 5′ end of exon 9and run through a portion of intron 9 to the cryptic poly A additionsignal in the MMTV genome. Intron nucleotide sequence is given in lowercase letter (corresponding to nucleotides 103–182 of SEQ ID NO: 35),MMTV sequences are underlined (corresponding to nucleotides 183–657 ofSEQ ID NO: 35), and dashes indicate nucleotide sequences of the intronwhich have been spliced out (corresponding to nucleotides 103–120 of SEQID NO: 35). The MMTV amino acid sequences are underlined (represented bySEQ ID NO: 36.) Dots correspond to amino acid residues encoded by RNAspecies 2 and 3 which are identical to those encoded by RNA species 1(see also amino acids 253–268 of SEQ ID NO: 4). The abbreviations foramino acids shown in FIGS. 9A and 9B are the same as those given in thelegend for FIG. 5.

FIG. 10 shows the genomic map of the human Int6 gene. As in the mousegenome, the human Int6 gene is composed of 13 exons (filled bars) wherethe nucleotide boundaries for each exon are presented in the Figure. Thelocation of a CA-repeat sequence in the seventh intron is also shown.

FIG. 11 shows the nucleotide sequence of primers (underlined, SEQ ID NO:31) complementary to the nucleic acid sequences (not underlined, SEQ IDNO: 32) flanking the CA-repeat in intron 7 of the human Int6 gene. Thedistance of the primers from the CA-repeat are presented as 40 and 138base pairs respectively and the number of CA-repeats (18) shown is thatfound in the wild-type Int6 gene. The upper nucleic acid sequences areshown in the 5′ to 3′ orientation and the lower nucleic acid sequencesare shown in the 3′ to 5′ orientation reading left to right.

FIG. 12 shows the results of Southern blots in which human DNA (lane 4),Chinese hamster-human somatic cell hybrid DNA containing only humanchromosome 6 (lane 1) or human chromosome 8 (lane 2) and mouse-humansomatic cell hybrid DNA which contains human chromosomes 3, 7, 8, 15 and17 (lane 3) were digested with Hind III and hybridized with human Int6cDNA.

FIG. 13 shows the results of PCR amplification of DNA from primary humanbreast tumor (T) and matching normal tissue (L, where L indicateslymphocyte) using labelled primers flanking the CA repeat sequence inintron 7 of the Int6 gene. The numbers above each blot representpatients from which tumor and matching normal tissue samples wereobtained. The arrow next to each blot indicates the complete loss, or asignificantly reduced signal, of one allele relative to the matchingnormal DNA.

FIG. 14 shows the nucleotide sequences bounding 12 of the 13 human Int6exons (SEQ ID NOs: 5–28). Nucleotide sequences of primers (underlined)complementary to nucleic acid sequences (not underlined) bounding eachexon are shown to the left and right of each exon. The upper nucleicacid sequences shown to the left and right of each exon are in the 5′ to3′ orientation (SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23 SEQ ID NO: 25 and SEQ ID NO: 27) while lower sequencesshown to the left and right of each exon are in the 3′ to 5′ orientation(SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ IDNO: 24 SEQ ID NO: 26 and SEQ ID NO: 28). When the sequences boundingeach exon do not begin at the intron-exon junction, the distance of thesequence from the junction is given in base pairs to the left or rightof each exon.

FIG. 15 shows the results of a Northern blot of total RNA (20 μg each)isolated from primary breast tumor (T) and matching normal (N) tissue.The RNA samples were denatured in the presence of formaldehyde, run on1% agarose gels containing formaldehyde, then transferred to a nylonmembrane and hybridized sequentially with a β-actin-probe and a humanInt6 cDNA probe.

FIG. 16 shows the results of a Northern blot of total RNA (20 μg each)isolated from human nonsmall cell lung carcinomas (54T and 55T) andmatching normal tissue (55N). Matching normal tissue for 54T was alsosurveyed for Int6 mRNA expression but the Northern blot for 54N is notshown. Northern blot analysis was carried out as described in FIG. 15.

DESCRIPTION OF INVENTION

The present invention discloses that mutational events associated withMMTV integration into the host cell genome in tumorigenesis occur in apreviously unknown gene designated Int6. This gene is located on mousechromosome 15. More specifically, the present invention relates to Int6gene and its corresponding cDNA. The region of mouse chromosome 15containing the Int6 is represented in the four overlapping lambda clones(designated 1–4 in FIG. 3) and comprises the entire Int6 gene. The Int6gene represents the wild-type Int6 gene.

The present invention is also directed to the full-length cDNAcorresponding to the murine Int6 gene. This cDNA sequence is set forthbelow as SEQ ID NO:1.

AACAAGCGCT CCTTTCCCCC GGCAAGATGG CGGAGTACGA 40 CCTGACTACT GCATCGCGCATTTTCTGGAT CGGCACCTGG 80 TCTTTCCGCT TCTTGAGTTT CTCTCTGTGA AAGAGATTTA 120TAATGAAAAA GAATTATTAC AAGGAAAATT AGATCTTCTT 160 AGTGATACCA ATATGGTGGACTTTGCTATG GATGTTTACA 200 AAAACCTTTA TTCTGATGAT ATCCCTCATG CTTTGAGAGA240 AAAAAGAACC ACAGTTGTTG CGCAGCTGAA ACAGCTCCAG 280 GCAGAAACAGAACCAATTGT GAAGATGTTT GAAGATCCAG 320 AAACTACAAG GCAGATGCAG TCAACCAGGGATGGCAGGAT 360 GTTATTTGAC TACCTGGCAG ACAAACATGG GTTTAGGCAA 400GAGTACTTAG ATACACTCTA CAGATACGCA AAATTCCAGT 440 ATGAGTGTGG AAATTACTCTGGAGCTGCAG AGTATCTTTA 480 CTTCTTTAGA GTTTTGGTCC CAGCAACAGA TAGAAATGCT520 TTAAGTTCGC TCTGGGGAAA ACTGGCCTCT GAAATCTTAA 560 TGCAGAATTGGGATGCAGCC ATGGAAGACC TTACTCGATT 600 AAAAGAAACC ATAGACAATA ATTCTGTGAGTTCTCCACTC 640 CAGTCTCTTC AGCAGCGAAC ATGGCTCATT CATTGGTCTC 680TATTTGTTTT TTTCAACCAT CCAAAGGGCC GTGATAACAT 720 TATTGATCTC TTCCTTTACCAACCACAGTA TCTTAATGCA 760 ATTCAGACAA TGTGTCCACA TATTCTACGC TATTTGACTA800 CTGCCGTCAT AACCAACAAA GATGTGCGGA AACGCCGGCA 840 GGTGCTGAAAGATCTGGTGA AAGTGATTCA ACAGGAGTCT 880 TACACATATA AAGACCCAAT TACAGAATTTGTTGAATGCC 920 TATATGTTAA CTTTGATTTT GACGGGGCTC AGAAAAAGCT 960GAGAGAATGT GAATCAGTGC TCGTGAATGA CTTCTTCCTG 1000 GTAGCGTGTC TGGAGGACTTCATTGAGAAT GCCCGTCTCT 1040 TCATATTTGA GACGTTTTGT CGTATCCACC AGTGTATCAG1080 CATTAATATG TTAGCAGATA AACTGAATAT GACTCCAGAA 1120 GAAGCTGAAAGATGGATTGT GAATTTGATT AGAAATGCGA 1160 GGTTGGATGC CAAGATTGAT TCTAAACTAGGTCATGTGGT 1200 AATGGGCAAC AATGCAGTCT CGCCCTACCA GCAAGTGATT 1240GAAAAGACCA AAAGCCTTTC TTTTAGAAGC CAAATGTTGG 1280 CCATGAATAT TGAAAAGAAACTTAATCAGA ACAGTAGATC 1320 AGAGGCTCCC AACTGGGCAA CCCAAGACTC TGGCTTCTAT1360 TAAAGGATTA TAAAGAAAAG AAGAAAAAGG AATAAGTGAA 1400 AGACACAGTAGCCATTGTGT ATAAAGGATG ACATACATTT 1440 TTAGAAGCAA TTAACATGTT TGCTACAAATTTTGGAGAAT 1480 TTGAATAAAA TTGGCTATGA TTAA 1504

The abbreviation used for the nucleotides are those standardly used inthe art.

The deduced amino acid sequence of the murine Int6 cDNA is shown as SEQID NO:2 below and starts at nucleotide 173 of SEQ ID NO:1 and extends1188 nucleotides.

Met Val Asp Phe Ala Met Asp Val Tyr Lys Asn Leu  1               5                  10 Tyr Ser Asp Asp Ile Pro His AlaLeu Arg Glu Lys          15                  20 Arg Thr Thr Val Val AlaGln Leu Lys Gln Leu Gln  25                  30                  35 AlaGlu Thr Glu Pro Ile Val Lys Met Phe Glu Asp             40                  45 Pro Glu Thr Thr Arg Gln Met Gln SerThr Arg Asp      50                   55                   60 Gly ArgMet Leu Phe Asp Tyr Leu Ala Asp Lys His                  65                  70 Gly Phe Arg Gln Glu Tyr Leu Asp Thr Leu Tyr Arg         75                  80 Tyr Ala Lys Phe Gln Tyr Glu Cys Gly AsnTyr Ser  85                  90                  95 Gly Ala Ala Glu TyrLeu Tyr Phe Phe Arg Val Leu             100                 105 Val ProAla Thr Asp Arg Asn Ala Leu Ser Ser Leu    110                 115                 120 Trp Gly Lys Leu Ala SerGlu Ile Leu Met Gln Asn                 125                 130 Trp AspAla Ala Met Glu Asp Leu Thr Arg Leu Lys         135                 140Glu Thr Ile Asp Asn Asn Ser Val Ser Ser Pro Leu145                 150                 155 Gln Ser Leu Gln Gln Arg ThrTrp Leu Ile His Trp             160                 165 Ser Leu Phe ValPhe Phe Asn His Pro Lys Gly Arg    170                 175                 180 Asp Asn Ile Ile Asp LeuPhe Leu Tyr Gln Pro Gln                 185                 190 Tyr LeuAsn Ala Ile Gln Thr Met Cys Pro His Ile         195                 200Leu Arg Tyr Leu Thr Thr Ala Val Ile Thr Asn Lys205                 210                 215 Asp Val Arg Lys Arg Arg GlnVal Leu Lys Asp Leu             220                 225 Val Lys Val IleGln Gln Glu Ser Tyr Thr Tyr Lys    230                 235                 240 Asp Pro Ile Thr Glu PheVal Glu Cys Leu Tyr Val                 245                 250 Asn PheAsp Phe Asp Gly Ala Gln Lys Lys Leu Arg         255                 260Glu Cys Glu Ser Val Leu Val Asn Asp Phe Phe Leu265                 270                 275 Val Ala Cys Leu Glu Asp PheIle Glu Asn Ala Arg             280                 285 Leu Phe Ile PheGlu Thr Phe Cys Arg Ile His Gln    290                 295                 300 Cys Ile Ser Ile Asn MetLeu Ala Asp Lys Leu Asn                 305                 310 Met ThrPro Glu Glu Ala Glu Arg Trp Ile Val Asn         315                 320Leu Ile Arg Asn Ala Arg Leu Asp Ala Lys Ile Asp325                 330                 335 Ser Lys Leu Gly His Val ValMet Gly Asn Asn Ala             340                 345 Val Ser Pro TyrGln Gln Val Ile Glu Lys Thr Lys    350                 355                 360 Ser Leu Ser Phe Arg SerGln Met Leu Ala Met Asn                 365                 370 Ile GluLys Lys Leu Asn Gln Asn Ser Arg Ser Glu         375                 380Ala Pro Asn Trp Ala Thr Gln Asp Ser Gly Phe Tyr385                 390                 395

The present invention also discloses the nucleotide sequence of thehuman homologue of the Int6 cDNA where the human Int6 cDNA sequence is89% homologous to the mouse sequence. Two overlapping recombinant clones(HINT6A and HINT6B) represent the 5′ and 3′ halves respectively of thehuman Int6 cDNA sequence. These two clones were deposited with theAmerican Type Culture collection (ATCC), 12301 Parklawn Drive,Parkville, Md. 20852 on Jan. 24, 1995 and have ATCC accession numbers97029 and 97030. The human Int6 cDNA sequence is shown below as SEQ IDNO:3.

ACTCCCTTTT CTTTGGCAAG ATGGCGGAGT ACGACTTGAC 40 TACTCGCATC GCGCACTTTTTGGATCGGCA TCTAGTCTTT 80 CCGCTTCTTG AATTTCTCTC TGTAAAGGAG ATATATAATG 120AAAAGGAATT ATTACAAGGT AAATTGGACC TTCTTAGTGA 160 TACCAACATG GTAGACTTTGCTATGGATGT ATACAAAAAC 200 CTTTATTCTG ATGATATTCC TCATGCTTTG AGAGAGAAAA240 GAACCACAGT GGTTGCACAA CTGAAACAGC TTCAGGCAGA 280 AACAGAACCAATTGTGAAGA TGTTTGAAGA TCCAGAAACT 320 ACAAGGCAAA TGCAGTCAAC CAGGGATGGTAGGATGCTCT 360 TTGACTACCT GGCGGACAAG CATGGTTTTA GGCAGGAATA 400TTTAGATACA CTCTACAGAT ATGCAAAATT CCAGTACGAA 440 TGTGGGAATT ACTCAGGAGCAGCAGAATAT CTTTATTTTT 480 TTAGAGTGCT GGTTCCAGCA ACAGATAGAA ATGCTTTAAG520 TTCACTCTGG GGAAAGCTGG CCTCTGAAAT CTTAATGCAG 560 AATTGGGATGCAGCCATGGA AGACCTTACA CGGTTAAAAG 600 AGACCATAGA TAATAATTCT GTGAGTTCTCCACTTCAGTC 640 TCTTCAGCAG AGAACATGGC TCATTCACTG GTCTCTGTTT 680GTTTTCTTCA ATCACCCCAA AGGTCGCGAT AATATTATTG 720 ACCTCTTCCT TTATCAGCCACAATATCTTA ATGCAATTCA 760 GACAATGTGT CCACACATTC TTCGCTATTT GACTACAGCA800 GTCATAACAA ACAAGGATGT TCGAAAACGT CGGCAGGTTC 840 TAAAAGATCTAGTTAAAGTT ATTCAACAGG AGTCTTACAC 880 ATATAAAGAC CCAATTACAG AATTTGTTGAATGTTTATAT 920 GTTAACTTTG ACTTTGATGG GGCTCAGAAA AAGCTGAGGG 960AATGTGAATC AGTGCTTGTG AATGACTTCT TCTTGGTGGC 1000 TTGTCTTGAG GATTTCATTGAAAATGCCCG TCTCTTCATA 1040 TTTGAGACTT TCTGTCGCAT CCACCAGTGT ATCAGCATTA1080 ACATGTTGGC AGATAAATTG AACATGACTC CAGAAGAAGC 1120 TGAAAGGTGGATTGTAAATT TGATTAGAAA TGCAAGACTG 1160 GATGCCAAGA TTGATTCTAA ATTAGGTCATGTGGTTATGG 1200 GTAACAATGC AGTCTCACCC TATCAGCAAG TGATTGAAAA 1240GACCAAAAGC CTTTCCTTTA GAAGCCAGAT GTTGGCCATG 1280 AATATTGAGA AGAAACTTAATCAGAATAGC AGGTCAGAGG 1320 CTCCTAACTG GGCAACTCAA GATTCTGGCT TCTACTGAAG1360 AACCATAAAG AAAAGATGAA AAAAAAAACT ATGAAAGAAA 1400 GATGAAATAATAAAACTATT ATATAAAGGG TGACTTACAT 1440 TTTGGAAACA ACATATTACG TATAAATTTTGAAGAATTGG 1480 AATAAAATTG ATTCATTTTA 1500

The deduced amino acid sequence of the human Int6 cDNA is shown below asSEQ ID NO:4 and starts at nucleotide 168 of SEQ ID NO:3 and extends 1188nucleotides.

Met Val Asp Phe Ala Met Asp Val Tyr Lys Asn Leu  1               5                  10 Tyr Ser Asp Asp Ile Pro His AlaLeu Arg Glu Lys          15                  20 Arg Thr Thr Val Val AlaGln Leu Lys Gln Leu Gln  25                  30                 35 AlaGlu Thr Glu Pro Ile Val Lys Met Phe Glu Asp             40                  45 Pro Glu Thr Thr Arg Gln Met Gln SerThr Arg Asp      50                  55                  60 Gly Arg MetLeu Phe Asp Tyr Leu Ala Asp Lys His                 65                  70 Gly Phe Arg Gln Glu Tyr Leu AspThr Leu Tyr Arg          75                  80 Tyr Ala Lys Phe Gln TyrGlu Cys Gly Asn Tyr Ser  85                  90                  95 GlyAla Ala Glu Tyr Leu Tyr Phe Phe Arg Val Leu            100                 105 Val Pro Ala Thr Asp Arg Asn Ala LeuSer Ser Leu     110                 115                 120 Trp Gly LysLeu Ala Ser Glu Ile Leu Met Gln Asn                125                 130 Trp Asp Ala Ala Met Glu Asp LeuThr Arg Leu Lys         135                 140 Glu Thr Ile Asp Asn AsnSer Val Ser Ser Pro Leu 145                 150                 155 GlnSer Leu Gln Gln Arg Thr Trp Leu Ile His Trp            160                 165 Ser Leu Phe Val Phe Phe Asn His ProLys Gly Arg     170                 175                 180 Asp Asn IleIle Asp Leu Phe Leu Tyr Gln Pro Gln                185                 190 Tyr Leu Asn Ala Ile Gln Thr MetCys Pro His Ile         195                 200 Leu Arg Tyr Leu Thr ThrAla Val Ile Thr Asn Lys 205                 210                 215 AspVal Arg Lys Arg Arg Gln Val Leu Lys Asp Leu            220                 225 Val Lys Val Ile Gln Gln Glu Ser TyrThr Tyr Lys     230                 235                 240 Asp Pro IleThr Glu Phe Val Glu Cys Leu Tyr Val                245                 250 Asn Phe Asp Phe Asp Gly Ala GlnLys Lys Leu Arg         255                 260 Glu Cys Glu Ser Val LeuVal Asn Asp Phe Phe Leu 265                 270                 275 ValAla Cys Leu Glu Asp Phe Ile Glu Asn Ala Arg            280                 285 Leu Phe Ile Phe Glu Thr Phe Cys ArgIle His Gln     290                 295                 300 Cys Ile SerIle Asn Met Leu Ala Asp Lys Leu Asn                305                 310 Met Thr Pro Glu Glu Ala Glu ArgTrp Ile Val Asn         315                 320 Leu Ile Arg Asn Ala ArgLeu Asp Ala Lys Ile Asp 325                 330                 335 SerLys Leu Gly His Val Val Met Gly Asn Asn Ala            340                 345 Val Ser Pro Tyr Gln Gln Val Ile GluLys Thr Lys     350                 355                 360 Ser Leu SerPhe Arg Ser Gln Met Leu Ala Met Asn                365                 370 Ile Glu Lys Lys Leu Asn Gln AsnSer Arg Ser Glu         375                 380 Ala Pro Asn Trp Ala ThrGln Asp Ser Gly Phe Tyr 385                 390                 395

The amino acid sequence of human and mouse Int6 proteins are identical.

Variations are contemplated in the cDNA sequences shown in SEQ ID NOs:1and 3 which will result in a DNA sequence that is capable of directingproduction of analogs of the proteins shown in SEQ ID NOs:2 and 4respectively. It should be noted that the DNA sequences set forth aboverepresents a preferred embodiment of the present invention. Due to thedegeneracy of the genetic code, it is to be understood that numerouschoices of nucleotides may be made that will lead to a DNA sequencecapable of directing production of the instant Int6 proteins or theiranalogs. As such, DNA sequences which are functionally equivalent to thesequence set forth above or which are functionally equivalent tosequences that would direct production of analogs of the Int6 proteinsproduced pursuant to the amino acid sequences set forth above, areintended to be encompassed within the present invention.

The term analog includes any protein or polypeptide having an amino acidresidue sequence substantially identical to a sequence specificallyshown herein in which one or more amino acid residues have beenconservatively substituted with a functionally similar residue. Examplesof conservative substitutions include, for example, the substitution ofone non-polar (i.e. hydrophobic) residue such as isoleucine, valine,leucine or methionine for another; the substitution of one polar (i.e.hydrophilic) residue for another, such as a substitution betweenarginine and lysine, between glutamine and asparagine, or betweenglycine and serine; the substitution of one basic residue such aslysine, arginine or histidine for another; or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another.

The phrase conservative substitution may also include the use of achemically derivatized residue in place of a non-derivatized residue.

Chemical derivative refers to an Int6 protein or polypeptide having oneor more residues chemically derivatized by reaction of a functional sidegroup. Examples of such derivatized molecules include, but are notlimited to, those molecules in which free amino groups have beenderivatized to form, for example, amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups. Free carboxyl groups may bederivatized to form salts, methyl and ethyl esters, or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzyllhistidine. Also included as chemicalderivatives are those proteins or peptides which contain one or morenaturally-occurring amino acid derivatives of the twenty standard aminoacids. For example, 4-hydroxyproline may be substituted for proline;5-hydroxylsine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

The nucleic acid sequences provided by the present invention are usefulas probes for a number of purposes. For example, they can be used asprobes in Southern hybridization to genomic DNA and in the RNaseprotection method for detecting point mutations. The probes can be usedto detect PCR amplification products. They may also be used to detectmismatches with the Int6 gene or mRNA using other techniques. Mismatchescan be detected using either enzymes (e.g., S1 nuclease), chemicals(e.g., hydroxylamine or osmium tetroxide and piperidine), or changes inelectrophoretic mobility of mismatched hybrids as compared to totallymatched hybrids. These techniques are known in the art. The probes arecomplementary to Int6 gene coding sequences, although probes to intronsare also contemplated. An entire battery of nucleic acid probes is usedto compose a kit for detecting alteration of wild-type Int6 genes. Thekit allows for hybridization to the entire Int6 gene. The probes mayoverlap with each other or be contiguous.

If a riboprobe is used to detect mismatches with mRNA, it iscomplementary to the mRNA of the human wild-type Int6 gene. Theriboprobe thus is an anti-sense probe in that it does not code for theInt6 protein because it is of the opposite polarity to the sense strand.The riboprobe generally will be labeled with a radioactive,colorimetric, or fluorometric materials, which can be accomplished byany means known in the art. If the riboprobe is used to detectmismatches with DNA it can be of either polarity, sense or anti-sense.Similarly, DNA probes also may be used to detect mismatches.

Nucleic acid probes may also be complementary to mutant alleles of Int6gene. These allele-specific probes are useful to detect similarmutations in other patients on the basis of hybridization rather thanmismatches. As mentioned above, the Int6 probes can also be used inSouthern hybridizations to genomic DNA to detect gross chromosomalchanges such as deletions and insertions. The probes can also be used toselect cDNA clones of Int6 genes from tumor and normal tissues. Inaddition, the probes can be used to detect Int6 mRNA in tissues todetermine if expression is altered as a result of mutation of wild-typeInt6 genes. Provided with the Int6 gene and the Int6 cDNA sequencesshown in SEQ ID NOs:1 and 3, design of particular probes is well withinthe skill of the ordinary artisan.

The present invention also relates to methods for detecting mutations ofthe Int6 gene in a subject.

For purposes of the present invention, subject means a mammal andmutation means inversion, translocation, insertion, deletion or pointmutation of the wild-type Int6 gene.

It is believed that many mutations found in tumor tissues will be thoseleading to altered expression of Int6 protein. However, mutationsleading to non-functional gene products can also lead to cancer. It isfurther understood that point mutations can occur in regulatory regions(e.g. promoter) or can disrupt proper RNA processing thus leading toreduced Int6 mRNA expression or loss of expression of the Int6 proteinrespectively.

The fact that integration of MMTV into the Int6 gene is observed inpreneoplastic mouse mammary lesions suggests that mutations of Int6 areinvolved in early events of cancer. The methods of detecting mutationsof the Int6 gene can therefore provide diagnostic and prognosticinformation. For example, detection of mutation of the Int6 gene in atumor may effect the course of treatment chosen by a clinician. Themethods of the present invention are applicable to any tumor in whichmutations of Int6 occur. Loss of expression of the Int6 gene has beenobserved in tumors of the lung and breast. Thus, these are tumors inwhich Int6 has a role in tumorigenesis. In addition, since Int6 isexpressed in all tissues tested including brain, heart, kidney, liver,ovaries, spleen and testes, mutations affecting the expression of theInt6 gene may contribute to neoplasia in these tissues too. Finally,since lung and breast tumors are derived from epithelial tissue, themethods of the invention may be useful in the detection of epithelialcell derived cancers such as nonsmall cell lung carcinomas.

It is further understood by one skilled in the art that the methods fordetection disclosed in the present invention can be used prenatally toscreen a fetus or presymptomatically to screen a subject at risk ofhaving cancer based on his/her family history.

In one embodiment of the invention, the method for detecting mutationsof the Int6 gene comprises analyzing the DNA of a subject for mutationsof the wild-type Int6 gene. For analysis of DNA, a biological specimenis obtained from the subject. Examples of biological specimens that canbe obtained for use in the present method, include, but are not limitedto, tissue biopsies and blood. Means for enriching a tissue preparationfor tumor cells are known in the art. For example, the tissue may beisolated from paraffin or cryostat sections. Cancer cells may also beseparated from normal cells by flow cytometry. These as well as othertechniques for separating tumor from normal cells are well known in theart. Alternatively, primary cell cultures can be established from tumorbiopsies using methods known to those of ordinary skill in the art.

The DNA isolated from the biological specimen can be analyzed formutations of the Int6 gene by a variety of methods including Southernblotting after digestion with the appropriate restriction enzymes(restriction fragment length polymorphism, RFLP) (Botstein, D. Amer. J.Hum. Genet. (1980) 69:201–205, denaturing gradient electrophoresistechnique (Myers, R. M., Nature (1985) 313:495–498), oligonucleotidehybridization (Conner, R. et al., EMBO J. (1984) 3:13321–1326), RNasedigestion of a duplex between a probe RNA and the target DNA (Winter, E.et al., Proc. Natl. Acad. Sci. U.S.A. (1985) 82:7575–7579), polymerasechain reaction (PCR) (Saiki, P. K. et al., Science (1988) 239:487–491;U.S. Pat. Nos. 4,683,195 and 4,683,202), ligase chain reaction (LCR)(European Patent Application Nos. 0,320,308 and 0,439,182), andPCR-single stranded conformation analysis (PCR-SSCP) (Orita, M. et al.,Genomics (1989) 5:874–879; Dean, M. et al. Cell (1990) 61:863–871).

In one preferred embodiment, Southern blot analysis can be used toexamine tumor or blood DNA for gross rearrangement of the Int6 gene. TheDNA to be analyzed via Southern analysis is digested with one or morerestriction enzymes. Following restriction digestion, resultant DNAfragments are separated by gel electrophoresis and the fragments aredetected by hybridization with a labelled nucleic acid probe (Southern,E. M. J. Mol. Biol. (1975) 98:503–517).

The nucleic acid sequence used as a probe in Southern analysis can belabeled in single-stranded or double-stranded form. Labelling of thenucleic acid sequence can be carried out by techniques known to oneskilled in the art. Such labelling techniques can include radiolabelsand enzymes (Sambrook, J. et al. (1989) in “Molecular Cloning, ALaboratory Manual”, Cold Spring Harbor Press, Plainview, N.Y.). Inaddition, there are known non-radioactive techniques for signalamplification including methods for attaching chemical moieties topyrimidine and purine rings (Dale, R. N. K. et al. (1973) Proc. Natl.Acad. Sci., 70:2238–2242; Heck, R. F. 1968) S. Am. Chem. Soc.,90:5518–5523), methods which allow detection by chemiluminescence(Barton, S. K. et al. (1992) J. Am. Chem. Soc., 114:8736–8740) andmethods utilizing biotinylated nucleic acid probes (Johnson, T. K. etal. (1983) Anal. Biochem., 133:126–131; Erickson, P. F. et al. (1982) J.of Immunology Methods, 51:241–249; Matthaei, F. S. et al. (1986) Anal.Biochem., 157:123–128) and methods which allow detection by fluorescenceusing commercially available products. The size of the probe can rangefrom about 200 nucleotides to about several kilobases. A preferred probesize is about 500 to about 2000 nucleotides. The probe may be derivedfrom introns or exons of the Int6 gene. Each of the nucleic acidsequences used as a probe in Southern analysis is substantiallyhomologous to the corresponding portion of the murine or human Int6genes where these genes have the cDNA sequences shown in SEQ ID NOs:1 or3 respectively. In a preferred embodiment, the probes are derived from ahuman Int6 gene having the Int6 cDNA sequence shown in SEQ ID NO:3. By“substantially homologous” is meant a level of homology between thenucleic acid sequence used as a probe and the corresponding sequence ofthe human or murine Int6 genes. Preferably, the level of homology is inexcess of 70%, most preferably in excess of 80%, with a particularlypreferred nucleic acid sequence being in excess of 90% homologous withthe sequence of the mouse or human Int6 genes.

Once the separated DNA fragments are hybridized to the labelled nucleicacid probes, the restriction digest pattern can be visualized byautoradiography and examined for the presence or absence of arestriction fragment length polymorphism (RFLP) associated with mutationof the Int6 gene.

In another preferred embodiment, genomic DNA may be analyzed formutations in the Int6 gene via PCR-SSCP. In this method, each of thepair of primers selected for use in PCR are designed to hybridize withsequences in the Int6 gene to permit amplification and subsequentdetection of mutations in the denatured amplification product vianon-denaturing polyacrylamide gel electrophoresis. In a preferredembodiment, primer pairs are derived from the human Int6 gene. In a morepreferred embodiment, primer pairs are derived from intronic sequenceswhich border the 5′ and 3′ ends of a given exon of the human Int6 gene.Examples of primer pairs permitting specific amplification of the exonsof the human Int6 gene include, but are not limited to,

ACCAATAAAGTTTTAGTGAGCACAG SEQ ID NO:5 GCGCCCAAAGACCCCCTCAC SEQ ID NO:6TTAATCAGTTTCTTTGGGGA SEQ ID NO:7 AGTTTCTAATGACAAAACTTAC SEQ ID NO:8TCTTCTGCATTTTTAATTAG SEQ ID NO:9 CAAAATTAAGACGAGTTTAC SEQ ID NO:10CTTATTTTGTTTCTGTGGCC SEQ ID NO:11 CATGACAACTTTAAAATATTTTT SEQ ID NO:12AATTACAATGGGGTTTTAAA SEQ ID NO:13 GAAGAACCAAGGGAATCCTA SEQ ID NO:14TTCAAGAGTATTCACAATAT SEQ ID NO:15 TGTGAAAAAGACGAACTCAC SEQ ID NO:16AGTTTTCTTTATCTCACCCT SEQ ID NO:17 CAATATATATTTTAGTTTTAC SEQ ID NO:18CCGTTGACTTATTTTTACAG SEQ ID NO:19 AAATAAAAATTCACACTTAC SEQ ID NO:20TTGTTGTATTTGTACATATAG SEQ ID NO:21 ATCAAATCACGGTGTTCTTAC SEQ ID NO:22AAAACTAAGTTTTTAGGCCC SEQ ID NO:23 ATAGCTAACATAATACTCAC SEQ ID NO:24TTCCCTGTGTTTCCTTTTAG SEQ ID NO:25 ATAGAAGATGTGTGGTCTTAC SEQ ID NO:26GATTTCTTTTTGCATATTTTAG SEQ ID NO:27 CAAGAAAACTGACAGCAAGA SEQ ID NO:28where SEQ ID NOs: 5 and 6 bound exon 1, SEQ ID NOs: 7 and 8 bound exon2, SEQ ID NOs: 9 and 10 bound exon 3, SEQ ID NOs: 11 and 12 bound exon4, SEQ ID NOs: 13 and 14 bound exon 5, SEQ ID NOs: 15 and 16 bound exon6, SEQ ID NOs: 17 and 18 bound exon 8, SEQ ID NOs: 19 and 20 bound exon9, SEQ ID NOs: 21 and 22 bound exon 10, SEQ ID NOs: 23 and 24 bound exon11, SEQ ID NOs: 25 and 26 bound exon 12, and SEQ ID NOs: 27 and 28 boundexon 13. Optimization of the amplification reaction to obtainsufficiently specific hybridization to Int6 gene sequences is wellwithin the skill in the art and is preferably achieved by adjusting theannealing temperature.

The primers of this invention can be synthesized using any of the knownmethods of oligonucleotide synthesis (e.g., the phosphodiester method ofAgarwal et al. 1972. Agnew. Chem. Int. Ed. Engl. 11:451, thephosphotriester method of Hsiung et al. 1979. Nucleic Acids Res. 6:1371,or the automated diethylphosphoramidite method of Beuacage et al. 1981.Tetrahedron Letters 22:1859–1862), or they can be isolated fragments ofnaturally occurring or cloned DNA. In addition, those skilled in the artwould be aware that oligonucleotides can be synthesized by automatedinstruments sold by a variety of manufacturers or can be commerciallycustom ordered and prepared. In one embodiment, the primers can bederivatized to include a detectable label suitable for detecting and/oridentifying the primer extension products (e.g., biotin, avidin, orradiolabeled dNTP's), or with a substance which aids in the isolation ofthe products of amplification (e.g. biotin or avidin).

In an alternative embodiment, primer pairs can be selected to hybridizeto mutant forms of the Int6 disease gene. The selected primer pairs willhybridize sufficiently specifically to the mutated gene sequences suchthat non-specific hybridization to wild-type Int6 gene sequences willnot prevent identification of the amplification product of the mutantgene sequence. Primer pairs which hybridize to mutations in the Int6gene sequence can be used to amplify specific mutant gene sequencespresent in the DNA of a biological sample.

The amplification products of PCR can be detected either directly orindirectly. Direct detection of the amplification products is carriedout via labelling of primer pairs. Labels suitable for labelling theprimers of the present invention are known to one skilled in the art andinclude radioactive labels, biotin, avidin, enzymes and fluorescentmolecules. The desired labels can be incorporated into the primers priorto performing the amplification reaction. A preferred labellingprocedure utilizes radiolabeled ATP and T4 polynucleotide kinase(Sambrook, J. et al. (1989) in “Molecular Cloning, A Laboratory Manual”,Cold Spring Harbor Press, Plainview, N.Y.). Alternatively, the desiredlabel can be incorporated into the primer extension products during theamplification reaction in the form of one or more labelled dNTPs. In thepresent invention, the labelled amplified PCR products can be analyzedfor mutations of the Int6 gene via separating the PCR products bynon-denaturing polyacrylamide gel electrophoresis, denaturingpolyacrylamide gel electrophoresis (PCR-SSCP) or via direct sequencingof the PCR-products.

In yet another embodiment, unlabelled amplification products can beanalyzed for mutations in the Int6 disease gene via hybridization withnucleic acid probes radioactively labelled or, labelled with biotin, inSouthern blots or dot blots. Nucleic acid probes useful in theembodiment are those described earlier for Southern analysis. In yetanother embodiment, detection of point mutations may be accomplished bymolecular cloning of the allele present in the tumor tissue using thecDNA sequences set forth in SEQ ID NOs:1 and 3 and sequencing thatallele using techniques well known in the art.

In a second embodiment, the method for detecting mutations of the Int6gene comprises analyzing the RNA of a subject for alterations inInt6-specific mRNA expression.

For the analysis of RNA by this method, RNA can be isolated from bloodor a tumor biopsy sample obtained from said subject where said tumorsinclude, but are not limited to, tumors of the breast and lung.

The RNA to be analyzed can be isolated from blood or tumor biopsysamples as whole cell RNA or as poly(A)⁺ RNA. Whole cell RNA can beisolated by methods known to those skilled in the art. Such methodsinclude extraction of RNA by differential precipitation (Birnbiom, H. C.(1988) Nucleic Acids Res., 16:1487–1497), extraction of RNA by organicsolvents (Chomczynski, P. et al. (1987) Anal. Biochem., 162:156–159) andextraction of RNA with strong denaturants (Chirgwin, J. M. et al. (1979)Biochemistry, 18:5294–5299). Poly(A)⁺ RNA can be selected from wholecell RNA by affinity chromatography on oligo-d(T) columns (Aviv, H. etal. (1972) Proc. Natl. Acad. Sci., 69:1408–1412). A preferred method ofisolating RNA is extraction of whole cell RNA by acid-phenol(Chomczynski et al. 1987).

The methods for analyzing the RNA for alterations in the pattern orlevel of Int6-specific mRNA expression include Northern blotting(Alwine, J. C. et al. (1977) Proc. Natl. Acad. Sci., 74:5350–5354), dotand slot hybridization (Kafatos, F. C. et al. (1979) Nucleic Acids Res.,7:1541–1522), filter hybridization (Hollander, M. C. et al. (1990)Biotechniques; 9:174–179), RNase protection (Sambrook, J. et al. (1989)in “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Press,Plainview, N.Y.), reverse-transcription polymerase chain reaction(RT-PCR) (Watson, J. D. et al. (1992) in “Recombinant DNA” SecondEdition, W.H. Freeman and Company, New York) and RT-PCR-SSCP. Onepreferred method is Northern blotting.

The nucleic acid sequence used as a probe for detecting Int6-specificmRNA expression is substantially homologous to SEQ. ID. NOs. 1 or 3. By“substantially homologous” is meant a level of homology between thenucleic acid sequence and the cDNA sequence of SEQ ID NOs:1 or 3.Preferably, the level of homology is in excess of 70% more preferably inexcess on 80%, with a particularly preferred nucleic acid sequence beingin excess of 90% homologous with the cDNA sequence shown in SEQ ID Nos.1 or 3.

In a second preferred embodiment, the RNA is analyzed for mutations inthe Int6 gene by RT-PCR-SSCP. Single stranded cDNA is prepared fromeither tumor total RNA or polyA⁺ enriched RNA using reversetranscriptase. In this method, each of the pairs of primers selected foruse in PCR of the resultant single-stranded cDNA are designed tohybridize with sequences in the Int6 cDNA which are an appropriatedistance apart (at least about 100–300 nucleotides) in the gene topermit amplification and subsequent detection of mutations in thedenatured amplification product via non-denaturing polyacrylamide gelelectrophoresis. Primer pairs which can specifically hybridize tooverlapping Int6 gene sequences can be derived from the Int6 genesequence. Primer pairs can be derived from the cDNA sequences set forthin SEQ ID NOs 1 and 3. In a preferred embodiment, the primers arederived from the human Int6 cDNA sequence shown in SEQ ID NO:3. Eachprimer of a pair is a single-stranded oligonucleotide of about 15 toabout 20 bases in length which is complementary to a sequence at the 3′end of one of the strands of a double-stranded target sequence. Eachpair comprises two such primers, one of which is complementary 3′ endand the other of which is complementary to the other 3′ end of thetarget sequence. The target sequence is generally about 100 to about 300base pairs long. Optimization of the amplification reaction to obtainsufficiently specific hybridization to the Int6 cDNA is well within theskill in the art and is preferably achieved by adjusting the annealingtemperature. Alternatively, the denatured RT-PCR products can beanalyzed for mutations of the Int6 gene via direct sequencing of theRT-PCR products.

Yet another preferred method of analysis is the RNase protection method,which is described in detail in Winter et al., Proc. Natl. Acad. Sci.USA, 82: p. 7575, (1985) and Meyers et al., Science, 230: p 1242,(1985). In the practice of the present invention the method involves theuse of a labeled riboprobe which is complementary to the human wild-typegene coding sequence. The riboprobe and either mRNA or DNA isolated fromthe tumor tissue are annealed (hybridized) together and subsequentlydigested with the enzyme RNase A which is able to detect mismatches in aduplex RNA structure. If a mismatch is detected by RNase A, it cleavesat the site of the mismatch. The riboprobe need not be the full lengthof the RNA or gene but can be a segment of either. If the riboprobecomprises only a segment of the Int6 RNA or Int6 gene it will bedesirable to use a number of these probes to screen the whole mRNAsequence for mismatches.

The present invention also encompasses recombinant proteins derived fromthe cDNAs shown in SEQ ID Nos. 1 and 3. Recombinant Int6 proteins can beproduced by recombinant DNA methodology known to one skilled in the art.Since the amino acid sequence of mouse (SEQ ID NO:2) and human (SEQ IDNO:4) Int6 proteins are identical, a suitable nucleic acid sequencecapable of encoding a protein comprising all or part of the amino acidsequence shown in SEQ ID NO:4 is the sequence shown in SEQ ID NO:3 or inSEQ ID NO:1. In a preferred embodiment, such a suitable nucleic acidsequence can be cloned into a vector capable of being transferred into,and replicated in, a host organism.

The vectors contemplated for use in the present invention include anyvectors into which a nucleic acid sequence as described above can beinserted along with any preferred or required operational elements, andwhich vector can then be subsequently transferred into a host organismand replicated in such organism. Preferred vectors are those whoserestriction sites have been well documented and which contain theoperational elements preferred or required for transcription of thenucleic acid sequence.

The “operational elements” as discussed herein include at least onepromoter, at least one operator, at least one leader sequence, at leastone terminator codon, and any other DNA sequences necessary or preferredfor appropriate transcription and subsequent translation of the vectornucleic acid. In particular, it is contemplated that such vectors willcontain at least one origin of replication recognized by the hostorganism along with at least one selectable marker and at least onepromoter sequence capable of initiating transcription of the nucleicacid sequence.

In construction of the cloning vector of the present invention, itshould additionally be noted that multiple copies of the nucleic acidsequence and its attendant operational elements may be inserted intoeach vector. In such an embodiment, the host organism would producegreater amounts per vector of the desired Int6 protein. The number ofmultiple copies of the DNA sequence which may be inserted into thevector is limited only by the ability of the resultant vector due to itssize, to be transferred into and replicated and transcribed in anappropriate host microorganism.

In another embodiment, restriction digest fragments containing a codingsequence for Int6 protein can be inserted into a suitable expressionvector that functions in prokaryotic or eukaryotic cells. By suitable ismeant that the vector is capable of carrying and expressing a completenucleic acid sequence coding for Int6 protein. Examples of expressionvectors that function in prokaryotic cells such as bacteria include, butare not limited to, T7 promoter based vectors and vectors for producingtrpE and lacZ fusions. Preferred expression vectors are those thatfunction in a eukaryotic cell. Examples of such vectors include but arenot limited to, vaccinia virus vectors, adenovirus, herpesviruses,baculovirus or mammalian type C retroviral vectors.

The selected recombinant expression vector may then be transfected intoa suitable eukaryotic cell system for purposes of expressing therecombinant protein. Such eukaryotic cell systems include, but are notlimited to cell lines such as human MCF10A or mouse HCll mammaryepithelial cells. One preferred eukaryotic cell system for use withbaculovirus vectors is SF21 ovarian cells from spodoptera frugyerda.

The expressed recombinant protein may be detected by methods known inthe art which include Coomassie blue staining and Western blotting usingsera containing anti-Int6 antibody.

In a further embodiment, the expressed recombinant protein can beobtained as a crude lysate or it can be purified by standard proteinpurification procedures known in the art which may include differentialprecipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis, affinity, andimmunoaffinity chromatography and the like. In the case ofimmunoaffinity chromatography, the recombinant protein may be purifiedby passage through a column containing a resin which has bound theretoantibodies specific for the Int6 protein.

In yet another embodiment, the present invention relates to peptidesderived from the Int6 amino acid sequences shown in SEQ ID Nos. 2 and 4where those skilled in the art would be aware that the peptides of thepresent invention, or analogs thereof, can be synthesized by automatedinstruments sold by a variety of manufacturers, can be commerciallycustom ordered and prepared, or can be expressed from suitableexpression vectors as described above. The term analog has beenpreviously described in the specification and for purposes of describingpeptides of the present invention, analogs can further include branchedor non-linear peptides. Preferred peptides are those that range fromabout 20 to about 24 amino acids length and are immunogenic. Asdescribed in Example 8 herein, mutation of the Int6 gene can result intruncation of the expressed Int6 protein. Based on previous studiesindicating that point-mutated ras proteins are processed in anappropriate manner to be targets for specific T cell-mediatedcytotoxicity (Tsang; K. Y. et al. (1994) Vaccine Research, 3:183–193;Takahashi, H. S.; et al. (1989) Science, 246:118–121, Jung, S.; et al.(1991) J. Exp. Med., 173:273–276, 1991; Peace, D. J.; et al. (1991) J.Immunol., 146:2059–2065; Fenton, R. G.; et al. (1993) J. Natl. CancerInst., 85:1294–1302; Gedde-Dahl, T., III; et al. (1992) Hum. Immunol.,33:266–274, and Gedde-Dahl, T., III; et al. (1992) Int. Immunol.,4:1331–1337), the mutated Int6 proteins may elicit a specific T-cellresponse via display on the tumor cell surface through the MHCapparatus.

The present invention therefore provides pharmaceutical compositionscomprising Int6 protein or peptides derived therefrom for use invaccines and in immunotherapy methods. When used as vaccines to protectmammals against cancer, the pharmaceutical composition can comprise asan immunogen cell lysate from cells transfected with a recombinantexpression vector or a culture supernatant containing the expressedprotein. Alternatively, the immunogen is a partially or substantiallypurified recombinant protein or a synthetic peptide.

The above compositions or formulations both for veterinary and for humanuse, comprise an immunogen as described above, together with one or morepharmaceutically acceptable carriers and optionally other therapeuticingredients. The carrier(s) must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. The formulations may convenientlybe presented in unit dosage form and may be prepared by any methodwell-known in the pharmaceutical art.

All methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1–2.0M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids which may be used eitheron their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11–10,000 parts by weight per part byweight of immunogen. If two or more stabilizers are to be used, theirtotal amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1–3.0 osmoles, preferably inthe range of 0.8–1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0–9.0, preferably within the range of 6–8. Informulating the immunogen of the present invention, anti-adsorptionagent may be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate theproteins, protein analogs or their functional derivatives, intoparticles of a polymeric material such as polyesters, polyamino acids,hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxy-methylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

The proteins of the present invention may be supplied in the form of akit, alone, or in the form of a pharmaceutical composition as describedabove.

Vaccination can be conducted by conventional methods. For example, theimmunogen can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. Further, the immunogen may or may notbe bound to a carrier to make the protein immunogenic. Examples of suchcarrier molecules include but are not limited to bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like.The immunogen can be administered by any route appropriate for antibodyproduction such as intravenous, intraperitoneal, intramuscular,subcutaneous, and the like. The immunogen may be administered once or atperiodic intervals until a significant titer of anti-Int6 antibody isproduced. The antibody may be detected in the serum using an immunoassayas described below.

In yet another embodiment, the present invention provides pharmaceuticalcompositions comprising nucleic acid sequence capable of directing hostorganism synthesis of an Int6 protein or of a peptide derived from theInt6 protein sequence. Such nucleic acid sequence may be inserted into asuitable expression vector by methods known to those skilled in the art.Expression vectors suitable for producing high efficiency gene transferin vivo include, but are not limited to, retroviral, adenoviral andvaccinia viral vectors. Operational elements of such expression vectorsare disclosed previously in the present specification and are known toone skilled in the art. Such expression vectors can be administeredintravenously, intramuscularly, subcutaneously, intraperitoneally ororally.

Whether the immunogen is an Int6 protein, a peptide derived therefrom ora nucleic acid sequence capable of directing host organism synthesis ofInt6 protein or peptides derived therefrom, the immunogen may beadministered for either a prophylactic or therapeutic purpose. Whenprovided prophylactically, the immunogen is provided in advance of thecancer or any symptom due to the cancer. The prophylactic administrationof the immunogen serves to prevent or attenuate any subsequent onset ofcancer. When provided therapeutically, the immunogen is provided at, orshortly after, the onset of cancer or any symptom associated with thecancer.

When the immunogen is a partially or substantially purified recombinantInt6 protein or an Int6 peptide, dosages effective to elicit an antibodyresponse against Int6 range from about 10 μg to about 1500 μg.

Dosages of Int6 protein-encoding nucleic acid sequence effective toelicit an antibody response against Int6 range from about 10 to about1000 μg.

The expression vectors containing a nucleic acid sequence capable ofdirecting host organism synthesis of an Int6 protein(s) may be suppliedin the form of a kit, alone, or in the form of a pharmaceuticalcomposition as described above.

The present invention further relates to a vaccine for immunizing amammal against cancer comprising Int6 protein or an expression vectorcapable of directing host organism synthesis of Int6 protein in apharmaceutically acceptable carrier.

In addition to use as vaccines and in immunotherapy, the abovecompositions can be used to prepare antibodies to Int6 protein. Toprepare antibodies, a host animal is immunized using the Int6 protein orpeptides derived therefrom or aforementioned expression vectors capableof expressing Int6 protein or peptides derived therefrom. The host serumor plasma is collected following an appropriate time interval to providea composition comprising antibodies reactive with the virus particle.The gamma globulin fraction or the IgG antibodies can be obtained, forexample, by use of saturated ammonium sulfate or DEAE Sephadex, or othertechniques known to those skilled in the art. The antibodies aresubstantially free of many of the adverse side effects which may beassociated with other anti-viral agents such as drugs.

The antibody compositions can be made even more compatible with the hostsystem by minimizing potential adverse immune system responses. This isaccomplished by removing all or a portion of the Fc portion of a foreignspecies antibody or using an antibody of the same species as the hostanimal, for example, the use of antibodies from human/human hybridomas.Humanized antibodies (i.e., nonimmunogenic in a human) may be produced,for example, by replacing an immunogenic portion of an antibody with acorresponding, but nonimmunogenic portion (i.e., chimeric antibodies).Such chimeric antibodies may contain the reactive or antigen bindingportion of an antibody from one species and the Fc portion of anantibody (nonimmunogenic) from a different species. Examples of chimericantibodies, include but are not limited to, non-human mammal-humanchimeras, rodent-human chimeras, murine-human and rat-human chimeras(Robinson et al., International Patent Application 184,187; TaniguchiM., European Patent Application 171,496; Morrison et al., EuropeanPatent Application 173,494; Neuberger et al., PCT Application WO86/01533; Cabilly et al., 1987 Proc. Natl. Acad. Sci. USA 84:3439;Nishimura et al., 1987 Canc. Res. 47:999; Wood et al., 1985 Nature314:446; Shaw et al., 1988 J. Natl. Cancer Inst. 80: 15553, allincorporated herein by reference).

General reviews of “humanized” chimeric antibodies are provided byMorrison S., 1985 Science 229:1202 and by Oi et al., 1986 BioTechniques4:214.

Suitable “humanized” antibodies can be alternatively produced by CDR orCEA substitution (Jones et al., 1986 Nature 321:552; verhoeyan et al.,1988 Science 239:1534; Biedleret al. 1988 J. Immunol. 141:4053, allincorporated herein by reference).

The antibodies or antigen binding fragments may also be produced bygenetic engineering. The technology for expression of both heavy andlight chain genes in E. coli is the subject the PCT patent applications;publication number WO 901443, WO901443, and WO 9014424 and in Huse etal., 1989 Science 246:1275–1281.

Alternatively, anti-Int6 antibodies can be induced by administeringanti-idiotype antibodies as immunogen. Conveniently, a purifiedanti-Int6 antibody preparation prepared as described above is used toinduce anti-idiotype antibody in a host animal. The composition isadministered to the host animal in a suitable diluent. Followingadministration, usually repeated administration, the host producesanti-idiotype antibody. To eliminate an immunogenic response to the Fcregion, antibodies produced by the same species as the host animal canbe used or the FC region of the administered antibodies can be removed.Following induction of anti-idiotype antibody in the host animal, serumor plasma is removed to provide an antibody composition. The compositioncan be purified as described above for anti-Int6 antibodies, or byaffinity chromatography using anti-Int6 antibodies bound to the affinitymatrix. The anti-idiotype antibodies produced are similar inconformation to the authentic Int6 antigen and may be used to preparevaccine rather than using an Int protein.

When used as a means of inducing anti-Int6 antibodies in an animal, themanner of injecting the antibody is the same as for vaccinationpurposes, namely intramuscularly, intraperitoneally, subcutaneously orthe like in an effective concentration in a physiologically suitablediluent with or without adjuvant. One or more booster injections may bedesirable.

For both in vivo use of antibodies to Int6 proteins and anti-idiotypeantibodies and for diagnostic use, it may be preferable to usemonoclonal antibodies. Monoclonal anti-Int6 antibodies or anti-idiotypeantibodies can be produced by methods known to those skilled in the art.(Goding, J. W. 1983. Monoclonal Antibodies: Principles and Practice,Pladermic Press, Inc., NY, N.Y., pp. 56–97). To produce a human-humanhybridoma, a human lymphocyte donor is selected. A donor known to havethe Int6 antigen may serve as a suitable lymphocyte donor. Lymphocytescan be isolated from a peripheral blood sample or spleen cells may beused if the donor is subject to splenectomy. Epstein-Barr virus (EBV)can be used to immortalize human lymphocytes or a human fusion partnercan be used to produce human-human hybridomas. Primary in vitroimmunization with peptides can also be used in the generation of humanmonoclonal antibodies.

Antibodies secreted by the immortalized cells are screened to determinethe clones that secrete antibodies of the desired specificity. Formonoclonal antibodies, the antibodies must bind to Int6 protein orpeptide. For monoclonal anti-idiotype antibodies, the antibodies mustbind to anti-Int6 antibodies. Cells producing antibodies of the desiredspecificity are selected.

The above described antibodies and antigen binding fragments thereof maybe supplied in kit form alone, or as a pharmaceutical composition for invivo use. When used as a pharmaceutical composition in immunotherapy,the antibodies or chimeric antibodies described herein may also becoupled to toxin molecules, radioisotopes, and drugs by conventionalmethods (Vitetta et al. (1991) in “Biologic Therapy of Cancer” De Vita VT, Hellman S., Rosenberg, S. A. (eds) J.B. Lippincott Co. Philadelphia;Larson, S. M. et al. (1991) in “Biological Therapy of Cancer” De Vita V.T., Hellman S., Rosenberg, S. A. (eds) J.B. Lippincott Co.,Philadelphia). Examples of toxins to which the antibodies may be coupledto include, but are not limited to, Ricin and pseudomonas endotoxin.Examples of drugs or chemotherapeutic agents include, but are notlimited to, adriamycin. Examples of radioisotopes, include, but are notlimited to, ¹³¹I. Antibodies covalently conjugated to the aforementionedagents can be used in cancer immunotherapy. The antibodies may also beused as an immunoaffinity agent to purify Int6 proteins, for therapeuticuses, and for diagnostic use in immunoassays as described below.

The present invention therefore relates to a third method for detectingmutations of the Int6 gene comprising:

analyzing the protein of a subject for alterations in Int6 proteinexpression.

For analysis of protein by this method, protein is obtained frombiological specimens such as tumor biopsy samples and the like. Theprotein can be obtained as a crude lysate or it can be further purifiedby methods known to one skilled in the art (Sambrook, J. et al. (1989)in “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor press,Plainview, N.Y.).

Crude protein lysate can be analyzed for Int6 protein by immunoassaysusing anti-Int6 antibody.

Immunoassays of the present invention may be a radioimmunoassay, Westernblot assay, immunofluorescent assay, enzyme immunoassay,chemiluminescent assay, immunohistochemical assay and the like. Standardtechniques known in the art for ELISA are described in Method inImmunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley andSons, 1980 and Campbell et al., Methods of Immunology, W.A. Benjamin,Inc., 1964, both of which are incorporated herein by reference. Suchassays may be a direct, indirect, competitive, or noncompetitiveimmunoassay as described in the art. (Oellerich, M. 1984. J. Clin. Chem.Clin. BioChem. 22:895–904).

Detection of the Int6 protein anti-Int6 antibody complex formed can beaccomplished by reaction of the complex with a secondary antibody suchas labelled anti-rabbit antibody. The label may be an enzyme which isdetected by incubating the complex in the presence of a suitablefluorimetric or colorimetric reagent. Other detectable labels may alsobe used, such as radiolabels, or colloidal gold, and the like. Thelabelled Int6 protein-anti-Int6 antibody complex is then visualized byautoradiography.

The present invention also includes a method for treating cancercomprising administering pharmaceutical compositions comprising an Int6protein, an expression vector containing nucleic acid sequence capableof directing host organism synthesis of an Int6 protein, or anti-Int6antibody in a therapeutically effective amount. When providedtherapeutically, the Int6 protein, Int6 protein-encoding expressionvector or anti-Int6 antibody coupled to radioisotopes, toxin moleculesor drugs is provided at (or shortly after) the onset of infection or atthe onset of any symptom of infection or disease caused by cancer. Thetherapeutic administration of the Int6 protein, Int6 protein-encodingexpression vector, or anti-Int6 antibody serves to attenuate theinfection or disease.

The present invention also includes another method for treating asubject having cancer comprising:

-   -   (a) immunizing a subject with an amount of Int6 protein or an        expression vector capable of directing host organism synthesis        of Int6 protein effective to elicit a specific T cell response;    -   (b) isolating said T cells from said immunized subject; and    -   (c) administering said T cells to said immunized subject or to        an unimmunized subject in a therapeutically effective amount.        T cells populations reactive against the Int6 protein may be        isolated from a peripheral blood sample or spleen cells of a        donor immunized with the Int6 protein. Epstein-Barr virus (EBV)        can be used to immortalize human lymphocytes or a human fusion        partner can be used to produce human-human hybridomas. Primary        in vitro immunization with Int6 protein can also be used in the        generation of T cells reactive to the Int6 protein.

T cells are cultured for about 7 to about 90 days (Yanelli, J. R. J.Immunol. Methods 139:1–16 (1991)) and then screened to determine theclones of the desired reactivity against the other peptide contained inthe immunogenic chimeric protein using known methods of assaying T cellreactivity; T cells producing the desired reactivity are thus selected.

The above described T cells may be used for in vivo use as treatment forindividuals afflicted with cancer by administering T cells to a mammalintravenously, intraperitoneally, intramuscularly or subcutaneously.Preferred routes of administration are intravenously orintraperitoneally.

The present invention also relates to a gene therapy method in which anexpression vector containing a nucleic acid sequence representing thewild-type Int6 gene is administered to a subject having a mutation ofthe Int6 gene. A nucleic acid sequence representing wild-type Int6 geneis that shown in SEQ ID No. 1 and SEQ ID No:3. Such nucleic acidsequence may be inserted into a suitable expression vector by methodsknown to those skilled in the art. Expression vectors suitable forproducing high efficiency gene transfer in vivo include retroviral,adenoviral and vaccinia viral vectors.

Expression vectors containing a nucleic acid sequence representingwild-type Int6 gene can be administered intravenously, intramuscularly,subcutaneously, intraperitoneally or orally. A preferred route ofadministration is intravenously.

The invention also provides a diagnostic kit for detecting mutations ofthe Int6 gene. This diagnostic kit comprises purified and isolatednucleic acid sequences useful as PCR primers in analyzing DNA or RNA formutations of the Int6 disease gene.

Any articles or patents referenced herein are incorporated by reference.The following examples are presented to illustrate various aspects ofthe invention but are in no way intended to limit the scope thereof.

EXAMPLES Example 1 Identification of a Novel Integration Site for theMMTV Genome

To identify new Int loci in mammary tumor DNAs that would not be biasedby selective inbreeding for a high cancer phenotype, a feral mousestrain derived from a single breeding pair of M. musculus trapped inCzechoslovakia (CZECH II) (Gallahan, D. and Callahan, R. (1987) J.Virol, 61:66–74) was utilized. The CZECH II mice, unlike high-incidenceinbred mouse strains, lack endogenous MMTV genomes but do contain aninfectious strain of MMTV that is transmitted congenitally through themilk (Callahan, R. et al. (1982) Proc. Natl. Acad. Sci. U.S.A.,79:4113–4117). In the CZECH II mice, several preneoplastic hyperplasticoutgrowth lines (HOGs) were developed which are stable, clonal-dominantand develop focal mammary tumors which sometimes metastasis to the lung.In order to detect the integration of MMTV proviral genome into genomicDNA of the HOGs or HOG-derived tumor or lung metastasis from a mousehaving a HOG-derived tumor, cellular DNA was isolated from a CZECH IIHOG line designated CZZ1 or from CZZ-1 derived malignant tumors or lungmetastasis and analyzed by Southern blotting as follows. 10 ug ofcellular DNA was digested by EcoRI, run on a 0.8% agarose gel,transferred to a nylon membrane and hybridized with MMTV LTR probe asdescribed in Gallahan, D. and Callahan R. (J. Virol (1987) 61:66–74. InBrief, before hybridization, filters were soaked for 4 to 24 hours at37° C. in a prehybridization solution containing 3×SSPE (1×SSPE is 180mM NaCl, 10 mM NaH₂PO₄ [pH 7.4], and 1 mM EDTA), 5×Denhardt solution(0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin),2.5% dextran sulfate, and 50% formamide. Denatured probe was added to asolution similar to the prehybridization solution except that itcontained 40% formamide. The mixture was added to the plastic bagcontaining prehybridized filters and incubated at 37° C. for 24 h. Thefilters were washed in stringent conditions involving three changes(each change done after 20 to 30 minutes of washing) of 0.1×SSC (1×SSCis 0.15 M NaCl plus 0.015 M sodium citrate) and 0.5% sodium dodecylsulfate at 65° C. The filters were exposed to Kodak XAR-5 C-ray filmovernight or for up to several days. The results of these Southern blotsare shown in FIGS. 1A and 1B. The results presented in FIG. 1A show thatDNA from one CZZ-1 HOG line contained three MMTV proviral genomes (sixEcoRI restriction fragments) integrated into the cellular DNA and thatthese proviral genomes were also present in primary tumors which aroseindependently from within the HOG. The results further demonstrated thatCZZ-1 was clonal-dominant population of preneoplastic cells becausethese six MMTV-related fragments were reproducibly found in the originaloutgrowth and in each succeeding transplant generation. Indeed, theclonal-dominant nature of the CZZ-1 HOG is illustrated by tumor 1262(lane 3, FIG. 1A) in which one complete proviral genome, correspondingto the 4.8 and 2.8 Kb EcoRI restriction fragments, has been lost. Theobservation that five out of the thirteen tumor DNAs analyzed in FIG. 1Aand five out of eleven independent metastatic lesions in the lung of themouse bearing tumor 4973 (FIG. 1B) contained additional integrated MMTVgenomes suggested that the additional integration events may havecontributed to tumor progression by activating (or inactivating)additional cellular genes. However, since none of the known commoninsertions sites (Wnt-1, Fgf-3, Int-3, Wnt-3, and Fgf-4) were rearrangedby MMTV in the CZZ-1 HOG, recombinant clones were obtained for each ofthe EcoRI host-MMTV junction restriction fragments in the CZZ-1 HOG andsubclones of the host sequences were used as probes to screen Southernblots of independent MMTV-induced mammary tumor DNAs for evidence ofMMTV-induced DNA rearrangements.

Using this approach, a probe consisting of the host sequences flankingone of the MMTV proviruses in the CZZ-1 HOG (i.e., the 3.0 Kb fragmentshown in FIG. 1A) was used to identify an Int6 common integration sitein the CZZ-1 HOG derived tumor 22 (FIG. 2A, lanes 2 and 3) andindependent mammary tumors 1139 (FIG. 2B, lanes 2 and 3) and 3144 (FIG.2C, lanes 2 and 3). In brief, cellular DNAs (10 μg) from each tumor DNAand from normal liver DNA (lane 1 in each panel) were digested withEcoRI, run on a 0.8% agarose gel, and then blotted onto a nylonmembrane. Lanes 1 and 2 of the blots in FIGS. 2A and 2B were hybridizedwith probe D which corresponds to host flanking sequences. Lane 2 ofFIGS. 2A and 2B was subsequently hybridized with MMTV gag sequences(Gallahan and Callahan (1987) J. Virol, 61:66–74) and the results areshown in lane 3 of FIGS. 2A and 2B. Lanes 1 and 2 in FIG. 2C werehybridized with probe C. Lane 2 was subsequently hybridized with MMTVenv sequences (Gallahan and Callahan (1987) J. Virol, 61:66–74) and theresult is shown in lane 3 of FIG. 2C. Hybridizations of the respectiveblots with the first probe were carried out as described earlier in thisExample (Gallahan and Callahan ((1987) J. Virol. 61:66–74). Where blotswere subsequently hybridized with a second probe, the blots werereprobed according to Gallahan and Callahan ((1987) J. Virol., 61:66–74)as follows. In brief, Genetran filters were stripped of probe DNA byplacing the filters in 100 ml of 0.4 N NaOH and incubated for 20 minutesat room temperature. This solution was then discarded and replaced with0.1 M Tris (pH 7.5)-0.1×SSC-0.5% sodium dodecyl sulfate solution toneutralize the filter. The filter was incubated for 15 minutes at roomtemperature, after which the neutralization solution was replaced andincubation was continued for another 15 minutes. The filter was thenprehybridized and hybridized as described earlier in this Example.Genetran filters treated in this way could be re-used at least fivetimes before noticeable. DNA loss.

The results show that MMTV-induced rearrangements (indicated by an arrowin FIGS. 2A–2C) were detected in the two independent MMTV-inducedmammary tumor DNAs (lanes 2 and 3 of FIGS. 2B and 2C) Of interest, ineach case where MMTV-induced rearrangements were detected, therearranged restriction fragment co-migrated with a fragment containingeither MMTV gag or env sequences. These results demonstrate that thetranscriptional orientation of the viral genome in each tumor was in thesame direction. Thus, the host sequences adjacent to the integrated MMTVgenome define a new common integration site from MMTV designated Int6.

Example 2 Isolation of the Murine Int6 Gene

To obtain recombinant genomic clones of the Int6 loci, a subclone of the3.0 kb EcoRI fragment (shown in FIG. 1A) containing the host flankingsequences (i.e. probe D, FIG. 3) was used to probe a lambda phagelibrary (Stratagene, LaJolla, Calif.) of genomic DNA from mouse strain129/sv. This genomic DNA contains wild-type Int6 DNA as determined bynucleotide sequence analysis. An additional three overlapping lambdaclones which span 47 kb of the Int6 locus have also been obtained usingprobes A–C (FIG. 3). Together, these four overlapping lambda clones spanthe murine Int6 gene as shown in FIG. 3. The gene is located centromericof the myc proto-oncogene on mouse chromosome 15. Further nucleotidesequence analysis of the genomic clones of the Int6 locus demonstratedthat the gene contains thirteen exons which span 34 Kb of genomic DNA asshown in FIG. 3.

Example 3 Expression of the Int6 Gene in CZECH II Mammary Tumors

To examine Int6 expression in CZECH II mammary tumors, total RNA (20 μg)prepared from the Int6 negative tumor (tumor 178) and from Int6 positivetumors having viral insertions in Int6 (22 and 1139) was denatured inthe presence of formaldehyde and run on a 1% agarose gel containingformaldehyde. The RNA was then transferred to a nylon membrane andhybridized with probe A (FIG. 3) as described in Gallahan, D. andCallahan, R. (1987) J. Virol., 61:66–74. The results of the Northernblots are shown in FIG. 4. In brief, hybridization with probe A detecteda 1.4 Kb species of RNA in each of three tumors and tumor 1139 alsocontained a 0.9 Kb species of RNA related to sequences in probe A. Theobservation that tumor 178 (FIG. 4) and several other MMTV inducedmammary tumors in which Int6 is not rearranged by the virus expressedthe 1.4 kb RNA species detected by probe A. This suggests that in tumors22 and 1139 the level of the Int6 gene expression is not an importantconsequence of viral integration in this locus.

Example 4 Isolation of Wild-Type Murine Int6 cDNA

In order to isolate a cDNA corresponding to wild-type 1.4 kb Int6 RNA, amurine cDNA library of an MMTV induced mammary tumor in which Int6 wasnot rearranged by MMTV was prepared using standard techniques (Sambrooket al (1989) in “Molecular Cloning, A Laboratory Manual”, Cold SpringHarbor Press, Plainview N.Y.) and was probed with probe A (probe A isthe XbaI fragment shown in FIG. 3). The nucleotide sequence of themurine Int6 cDNA was determined and is shown in FIG. 5. Translation ofthe 1.4 kb species of Int6 RNA revealed an open reading frame whichencodes a 43.5 kilodalton protein. As shown in FIG. 5, this proteincontains two potential N-glycosylation site motifs (Gavel, Y. and vonHeijne, G. (1990) Protein Eng., 3:433–442) as well as potentialphosphorylation site motifs for cyclic AMP/cyclic GMP-dependent proteinkinase (Glass, D. B. et al (1986) J. Biol. Chem., 261:2987–2993),protein kinase C (Kishimoto, A. et al (1985) J. Biol. Chem.,260:12492–12499), tyrosine kinases (Hunter, T. and Cooper, J. A. (1985)Ann. Rev. Biochem, 54:897–930) and casein kinase II (Pinna, L. A. (1990)Biochim Biophys Acta, 1054:267–284).

Example 5 Conservation of the Int6 cDNA Across Species

Since all of the genes whose expression has been affected or altered byMMTV integration in mouse mammary tumors have been highly conservedthrough evolution (Dickson, C. and Peters, G. (1987) Nature 326:883;Rijsewizk, K. F. et al (1987) Cell, 50:649–657; Robbins, J. et al (1992)J. Virol., 66:2594–2599), the conservation of the Int6 gene in genomicDNA of different species was examined via Southern blot analysis.Cellular DNAs (10 μg each) from C. elegans, Drosophila, Xenopus,chicken, mouse, and human were digested with BamHI, run on a 0.8%agarose gel, transferred to a nylon membrane and hybridized overnitewith Int6 cDNA in 3×SSPE (1×SSPE is 180 mM BaCl, 10 mMNaH₂PO₄ (pH 7.4,and 1 mM EDTA), 5×Denhardt solution (0.02% Ficoll, 0.02%polyvinylpyrrolidone, 0.02% bovine serum albumin), 5% dextran sulfateand 2% SDS (sodium dodecyl sulfate) at 65° C. After hybridization, theblot was washed in stringent condition involving three changes (eachchange done after 20 to 30 minutes of washing) 0.5×SSC (1×SSC is 0.15 MNaCl plus 0.015 M sodium citrate) plus 0.5% SDS at 65° C. Exposure toKodak XAR-5 film was for three days. As shown in FIG. 6, Int6 relatedsequences can be detected in all the eukaryotic species examined.Restriction fragments of yeast DNA which contain Int6 related sequenceswere also detected by Southern blot analysis (data not shown).

In addition, the nucleotide sequence of a cDNA clone of the Drosophilahomolog of Int6 has been determined (data not shown) and the deducedamino acid sequence of the Drosophila Int6 protein is 60% identical tothe human/mouse deduced amino acid sequences. Taken together, theseresults demonstrate an extensive evolutionary conservation of the Int6gene which is indicative of Int6 serving a basic life function.

Example 6 Detection of Int6-specific mRNA Expression in Target Tissues

Having detected the 1.4 Kb Int6 RNA species in a tumor in which the genewas not rearranged by MMTV (i.e., tumor 178, FIG. 4) normal adulttissues and embryos at different stages of development were surveyed forInt6 RNA expression via Northern analysis as follows. Total RNAs (10 μgeach) was prepared from the indicated tissues. The RNAs were denaturedin the presence of formaldehyde and run on 1% agarose gels containingformaldehyde. The RNA samples were then transferred to a nylon membraneand hybridized sequentially with a β-actin probe and then an Int6 cDNAprobe under conditions described in Gallahan and Callahan (J. Virol.,61:66–74 (1987)). As shown in FIG. 7, Int6 RNA is expressed in all adulttissues tested, including the mammary gland, and expression of Int6 RNAin embryos was detected as early as day eight of development.

Example 7 Detection of Int6-specific mRNA Expression in Target Tissues

To detect Int6-specific mRNA expression, Random primed cDNA is preparedusing total RNA from the various tissues. A 700 bp fragment of Int6murine cDNA is amplified by PCR using the primers shown as SEQ ID NO. 29TGTCCACATATTCTACGCTA and SEQ ID NO. 30 TGTATGTCATCCTTTATACA. Theconditions of the PCR are: 30 cycles of denaturation at 94° C. for 1minute, annealing at 55° C. for 1 minute, and extension at 72° C. for 2minutes. After the last cycle there is an additional extension time of 2minutes. The PCR product is run on a 1.0% agarose gel. The RT-PCRproducts are detected by photographing the gel following staining withethidium bromide.

Example 8 Rearrangement of Int6 by MMTV Leads to the Expression of NovelSpecies of Int6 RNA in Mammary Tumors

Inspection of the mapping data for the murine Int6 gene as shown in FIG.3 reveals that all of the viral integration events occur within intronsof the Int6 gene and that the transcriptional direction of theintegrated viral genome is in the opposite orientation to that of theInt6 gene. There are at least four possible scenarios for the role MMTVplays at the Into locus. In the first, the Int6 gene is in fact not thetarget gene on which MMTV acts. At the Wntl, Fgf-3, and Fgf-4 loci(Dickson, C., et al. (1984) Cell. 37:529–536; Nusse, R., et al. (1982)Cell. 31:99–109; Peters, G., et al. (1989) Proc. Natl. Acad. Sci. USA.86:5678–5682), MMTV integration sites cluster around the target gene inan ordered fashion and the transcriptional orientation of the integratedviral genomes 5′ of the target gene are in the opposite direction tothat of the target gene whereas those 3′ of the target gene are in thesame transcriptional orientation. Further, based on published reports,MMTV integration sites are within 15 kb of the particular target gene.However, since no evidence was found for the activation of expression ofRNA corresponding to sequences up to 13 kb 3′ of the Int6 gene, thelocation of the putative target gene would have to be more than 24 kbfrom the integrated MMTV genome in CZZ-1 HOG and more than 30 kb intumor 1139 (see FIG. 3), thereby making this possibility much lesslikely.

A second possible consequence of MMTV integration into the Int6 gene isthe activation of expression of a new chimeric RNA transcript of Int6initiated by the 3′ LTR of the integrated viral genome. An analogoussituation occurs in MMTV-induced rearrangements of Int-3 (Robbins, J.,et al. (1992) J. Virol., 66:2594–2599) except in the case of Int6, thiswould result in the expression of Int6 anti-sense RNA since thetranscriptional orientation of the viral genome is opposite to that ofInt6. Such a result would represent a trans-dominant mutation that wouldinactivate the expression of both alleles. However, using a variety oftechniques, MMTV-induced Int6 antisense RNA expression has not beendetected.

A third possibility assumes that the Int6 gene is the target for MMTVintegration within the Int6 locus. In this case the viral insertionwould disrupt the expression of one allele and reveal the presence of aspontaneous recessive mutation in the other allele. To examine thispossibility the nucleotide sequence of cDNA corresponding to thenon-rearranged allele of Int6 in tumor 22 and tumor 1139 was determinedand in both cases no mutation was found.

The fourth and more plausible scenario is that MMTV integration into theInt6 gene causes the expression of a biologically activated gene product(like Int3, (Robbins, J., et al. (1992) J. Virol., 66:2594–2599)) or adominant-negative gene product either of which deregulates the normalcontrol of mammary epithelial cellular growth leading to hyperplasia ofthe affected mammary epithelial cells. This would create a premalignantepithelial cell population with which mammary tumors could subsequentlydevelop.

To test whether integration of MMTV into the Int6 gene produces alteredRNA species, the nucleotide sequence of cDNA clones of Int6 RNA fromtumor 1139 and tumor 22. In each case transcription of the rearrangedallele resulted in the expression of a chimeric RNA species whichterminated at a cryptic transcription stop signal in the reverse U3portion of the MMTV LTR (FIG. 8). Of interest, a similar cryptictranscription termination signal has previously been shown to be activein certain MMTV induced rearrangements Fgf-3(Clausse, N., (1993)Virology, 194:157–165).

FIGS. 9A and 9B show the nucleotide-sequences of Int6-MMTV LTR RNAspecies detected in tumors 1139 (FIG. 9A) and 22 (FIG. 9B). In FIG. 9A,two RNA species from the rearranged allele (Figure A, 900 bp and 965 bp)were detected. The RNA species correspond to the 900 bp RNA speciesdetected by Northern blot analysis in FIG. 4. In one RNA species exon 5was spliced to the end of the U5 portion of the MMTV LTR and in theother species splicing occurred at a cryptic splice acceptor site inintron 5. Similarly, FIG. 9B, CZZ-1 there were three chimeric RNAspecies in which exon 9 was spliced to one of three different crypticsplice acceptor sites in intron 9. Since the size of the chimeric tumorRNA species is similar to that of the normal Int6 RNA, they wentundetected in the Northern blot analysis of tumor 22 RNA FIG. 4.

Translation of the rearranged Int6 RNA species into putative proteinsrevealed that from all five species, the product is a truncated chimeraof the Int6 amino acid sequences linked to novel amino acid sequencesencoded by an Int6 intron and/or reverse MMTV LTR nucleotide sequences.The presence of the 965 bp RNA species in tumor 1139 suggests thattruncation of the Int6 gene product is an important consequence of MMTVintegration.

Example 9 Isolation of the Human Int6 Gene

Mouse Int6 cDNA was used to probe a cDNA library of human lung RNA inlambda phage (Clontech Inc.) and using primers specific for the humanInt6 cDNA (SEQ ID NO:3) cloned from the human lung cDNA library, a P1phage library of human genomic DNA was screened by PCR for anInt6-related clone by Genome Systems Inc.(St. Louis, Mo.). Using thisapproach, a P1 phage clone and two lambda clones containing over 50 kbof human genomic DNA were obtained. Nucleotide sequence analysis ofthese clones revealed that the human Int6 gene is organized as shown inFIG. 10. As in the mouse genome, the human Int6 gene is composed of 13exons. The human Int6 gene also contains a CA-repeat sequence in theseventh intron (FIG. 10).

Example 10 Size Polymorphism of the CA Reseat Sequence Contained inIntron 7

Primers complementary to the nucleic acid sequences flanking theCA-repeat in intron 7 of the human Int6 gene (FIG. 1) and having thesequences shown in SEQ ID NO:31 GTGAAAATGACATGAAATTTCAG and SEQ ID NO:32TGCAGTGTGACAATATGGGC were used to PCR amplify the portion of the Int6gene containing the CA-repeat in human genomic DNA. The conditions ofthe PCR were: 30 cycles of denaturation at 94° C. for one minute,annealing at 55° C. for one minute, and extension at 72° C. for twominutes. After the last cycle, there was an additional extension time oftwo minutes. Separation of the PCR products via non-denaturing 6%polyacrylamide gel electrophoresis revealed that 46 of the 84 individualDNAs analyzed were heterozygous (informative) for different sizealleles.

Example 11 Chromosomal Localization of the Human Int 6 Gene

To determine the chromosomal localization of the human Int6 gene,Southern blot analysis of human DNA and DNA isolated from rodent-humansomatic cell hybrids containing individual human chromosomes wasperformed. In brief, rodent-human somatic cell hybrid DNA or human DNAwas digested with HindIII and hybridized with human Int6 cDNA at 37° C.for 24 hours in 3×SSPE, 5×Denhardt solution, 2.5% dextran sulfate and40% formamide. The blot was then washed under stringent conditionsinvolving three changes (each change done after twenty to thirtyminutes) of 0.1×SSC and 0.5% SDS at 65° C. The results of this Southernblot are shown in FIG. 12 where lanes 1 and 2 contain Chinesehamster-human somatic cell hybrid DNA containing human chromosome 6(lane 1) or 8 (lane 2), lane 3 contains DNA from mouse-human somaticcell hybrids containing human chromosomes 3, 7, 8, 15 and 17 and lane 4contains human genomic DNA. The 23 kb fragment shown in lane 4 containsan Int6-related pseudogene and the coding sequences of the Int6 gene aredefined by the 4.5, 3.5 and 3.0 kb fragments shown in lane 4. Theresults show that while the 23 kb fragment was detected only in lane 1(hybrids containing human chromosome 6), the 4.5 and 3.5 kb fragmentswere detected in lane 2 (hybrids containing human chromosome 8). Inaddition, the 3.0 kb human fragment (see lane 4) was also detected inboth lanes 1 and 2. However, the detection of the 3.0 kb fragment inlane 3 confirmed that the 3.0 kb fragment contains human Int6-relatedsequence rather than Chinese hamster sequence since mouse DNA was shownnot to contain an Int6-related 3.0 kb Hind III fragment. These resultsdemonstrate that the human Int6 gene is located on chromosome 8. Thischromosomal localization was confirmed and further refined to chromosome8q22-q24 by linkage analysis using the CA-repeat polymorphism describedin Example 10.

Example 12 Detection of Mutations in the Int6 Gene in Human Breast TumorDNA Resulting in a Loss of Heterozygosity

Based on the results in MMTV-induced mouse mammary tumors presentedearlier, DNA from human breast cancer biopsies was analyzed to determinewhether the Int6 gene might be a target for mutation during malignantprogression in human breast cancer. In brief, DNA from primary humanbreast tumor and from matching normal tissue of 40 individuals that wereinformative at the Int6 CA-repeat sequence were analyzed for evidence ofloss of heterozygosity (LOH) in the tumor DNA via the PCR methodutilized in Example 10. In eleven of these tumors (25%), there waseither a complete loss or a significantly reduced signal of one allelerelative to the matching normal DNA. Five of these eleven tumor DNAs (T)and their matching normal samples (L) are shown in FIG. 13. Theseresults show that Int6 is a target for mutation during malignantprogression in human breast cancer. Further, since Int6 gene expressionhas been detected in all tissues analyzed (see FIG. 7), mutation of theInt6 gene may occur during progression of cancers found in othertissues.

Example 13 Analysis of the Human Int6 Gene for Mututions by PCR-SSCPUsing Primer Pairs Derived from Sequences Bounding Exons of the Int6Gene

Primer pairs complementary to nucleic acid sequences bounding 12 of the13 human Int6 exons (FIG. 14) are shown as SEQ ID NOs:5–28. Using primerpairs selected from SEQ ID NOs:5–28, each of the exons of the Int6 geneexcept exon 7 were amplified from the eleven primary breast tumor DNAshaving loss of heterozygosity at the Int6 gene (see Example 12) and fromDNA of matching normal tissue samples by PCR and the PCR products wereanalyzed for single-stranded conformation polymorphism (SSCP) viadenaturing polyacrylamide gel electrophoresis. Exon 7 was analyzed byhybrid mismatch methodology using the portion of human Int6 cDNAcorresponding to exon 7. Analysis by both single strand conformationpolymorphism analysis (SSCP) and hybrid mismatch methodologies of theprimary breast tumor DNAs having loss of heterozygosity at the Int6 genedetected no point mutations in the remaining allele (data not shown).However, the promoter and intron regions of the remaining allele werenot analyzed for mutations. Of course, if mutations of the promoter,intron and/or coding regions of the Int6 gene were detected in tumorsamples, these mutations could be confirmed by nucleotide sequenceanalysis of the variant allele in the tumor-and-matching-normal tissueDNA.

Example 14 Loss of Expression of Int6 mRNA in Human Breast and LungTumor Samples

Total RNA isolated from 36 malignant human breast tumors, 2preneoplastic lesions (high grade dysplasia) and matching normal tissuewere surveyed for Int6 mRNA expression via Northern blot analysis usingsequential hybridization with β-actin and human Int6 cDNA probes underconditions described in Gallahan and Callahan (J. Virol. 61:66–74(1987)). Of these 38 samples, 14 (37%) exhibited significantly reducedor nondetectable Int6 mRNA expression as compared with matching normaltissue samples.

Of these 14 cases, 8 were invasive ductal carcinomas (IDC), 2 werecomedocarcinomas (IDC with a prevalent intraductal component), 2 werelobular carcinomas and 2 were the preneoplastic lesions. Arepresentative Northern blot is shown in FIG. 15.

In a separate study, Int6 mRNA expression in 47 nonsmall cell lungcarcinomas (NSCLC) and matching normal tissues was surveyed via Northernanalysis of 10 μg total RNA as described above in this Example. Arepresentative Northern blot is shown in FIG. 16 and the results of theNorthern analyses of the 47 NSCLC samples are summarized in Table 1.

TABLE 1 ASSOCIATIONS BETWEEN LOSS OF EXPRESSION OF Int6 AND CLINICALPARAMETERS REDUCED OR NONDETECTABLE NORMAL TOTAL HISTOTYPE EXPRESSIONEXPRESSION SAMPLES P VALUE SQUAMOUS CELL 3(14%) 18 21 ADENOCARCINOMA8(67%) 4 12 0.003 BRONCHIOLOALVEOLAR 2(33%) 4 6

Of interest, 14 of 47 NSCLCs showed loss of Int6 mRNA expression andloss of Int6 mRNA expression was observed to have a highly significantassociation (P=0.003) with a particular histologic subtype (Table 1,adenocarcinoma of NSCLC).

The results presented in this Example are consistent with the conclusionthat loss of Int6 expression is an early event in human breast cancerand a contributing factor in other neoplasias such as those of the lung.

Example 15 Characterization of the Murine Int6 Protein

The Int6 gene encodes a 50 kD protein with three potential translationstart sites (at 27 bp, 174 bp and 189 bp of the mouse sequence). Each ofthese polypeptides were detected as products of in vitro translation ofInt6 RNA in the rabbit reticulocyte system. Rabbit polyclonal sera tosynthetic peptides corresponding to amino acid residues 57–71 (peptide47) and 262–281 (peptide 20) of mouse Int6 were prepared and used toimmunoprecipitate the Int6 in vitro translation products. Thisimmunoprecipitation was competed by the corresponding peptide. Westernblot analysis of protein extracts of adult mouse tissues (brain, lung,kidney, muscle, heart and mammary gland), using antibodies to peptide 20(rAB 20), detected a major 40 KD polypeptide as well as a triplet of 80KD polypeptides. Reaction with these polypeptides was competed withpeptide 20. Of interest, expression of a 30 KD Int6 related polypeptidewas also observed in Western blots of salivary gland extracts. It isbelieved that the 40 KD polypeptide may represent a cleavage product ofthe 50 KD precursor and that the 80 KD cross reacting polypeptide mayrepresent dimers of the 40 KD polypeptides.

In addition, cell fractionation studies and immunofluorescence studiesusing antibodies to the above peptides have demonstrated that Int6 isprimarily localized to the cytoplasm. Moreover, in immunofluorescencestudies of mouse embryos, the Int6 protein was localized to the golgiapparatus.

Finally, Int6 protein, which contains three candidate protein kinase C(PKC) phosphorylation sites, was expressed in bacteria using the QUIAExpress Type 4 vector (Quiagen, Chatsworth Calif.), purified and shownto be phosphorylated by PKC (data not shown).

1. A purified antibody or fragment thereof that specifically binds SEQID NO: 4, or a fragment of about 20 to about 24 consecutive amino acidsof SEQ ID NO:
 4. 2. The purified antibody of claim 1, wherein theantibody is a monoclonal antibody.
 3. The purified antibody of claim 2,wherein the antibody is a humanized antibody.
 4. The purified antibodyof claim 1, covalently linked to a toxin, radionucleotide, detectablelabel or drug.
 5. The isolated antibody of claim 4, covalently linked toa toxin.
 6. The purified antibody of claim 4, covalently linked to aradionucleotide.
 7. The purified antibody of claim 4, covalently linkedto a detectable label.
 8. The purified antibody of claim 1, wherein theantibody is a polyclonal antibody.
 9. The purified antibody of claim 1,wherein the antibody specifically binds to a polypeptide encoded by acDNA deposited as ATCC Accession No. 97209 or as ATCC Accession No.97030.
 10. A kit, comprising a container containing the purifiedantibody of claim
 1. 11. The kit of claim 10, further comprising acontainer comprising a second antibody that specifically binds thepurified antibody that specifically binds SEQ ID NO:4, wherein thesecond antibody comprises a label.
 12. An isolated antibody thatspecifically binds amino acids 262–281 or amino acids 57–71 of SEQ IDNO: 4.